The roles of iron and light as limiting and colimiting factors for phytoplankton growth in subsurface chlorophyll maxima (SCMs) were investigated in mesotrophic to oligotrophic waters of the Southern California Bight and the eastern tropical North Pacific using microcosm manipulation experiments. Phytoplankton responses indicative of iron-light colimitation were found at several SCMs underlying macronutrient-limited surface waters in the eastern Pacific. Iron additions led to a shift in the size and taxonomic structure of the phytoplankton community, where large diatoms dominated what was formerly a diverse community of relatively small phytoplankton. The strongest and most ubiquitous responses of diatoms to iron addition were found under elevated light conditions, indicating that iron availability may have the greatest potential to affect SCM phytoplankton communities when light levels increase rapidly, such as during eddy events or with strong internal waves. The results show that iron influences phytoplankton community structure at SCMs, which would have consequences for nutrient cycling and carbon export within the lower euphotic zone.